In the study of diseases, cell or tissue heterogeneity has limited the information available from analysis of biological samples. It has become increasingly important to be able to investigate mRNA expression patterns within specific cell populations at a specific physiological state.
Histochemical approaches have been applied to identify specific cell populations within a biological sample. See, e.g., Okuducu et al. (2003), International Journal of Molecular Medicine 11:449–453. Such approaches include, e.g., techniques of immunohistochemistry that detect proteins, in-situ hybridization that measures messenger RNA, and fluorescence in-situ hybridization (FISH) that detects changes in DNA. For example, histochemical methods may be used to identify a cell type, e.g., microglia in the brain as identified by the expression of complement receptor 3 (Graeber et al. (1988), J Neurosci Res 21:18–24), or to identify a specific cell state, e.g., cellular activation stage as shown by c-Fos expression (Sagar et al. (1988), Science 240:1328–1331).
Following the identification of specific cell populations at a specific physiological state by histochemical analysis, mRNA expression patterns of cells of interest can be analyzed by traditional in-situ hybridization, which is often limited to detecting the expression of only one or very few genes. Alternatively, bioarray gene profiling can be performed. Cells of interest can first be isolated via techniques such as laser capture microdissection (LCM). mRNA can be extracted, amplified, and reverse transcribed from the isolated cells. The resulting cDNAs can be hybridized to a gene microarray chip. The resultant pattern of hybridized nucleic acid provides information regarding the genetic profile of the sample tested. This approach can be used to examine the expression of multiple genes within individual cells or tissues, and can be combined with other studies such as electrophysiological, pharmacological and anatomical (retrograde labeling) studies.
Analyses of gene expression patterns of an identified cell or tissue type make it possible to directly correlate gene expression with functional changes and lesion morphology at the target cells or tissue. Results from such analyses can provide important information on the effects of a drug within a biological test system and help to elucidate mechanisms of drug-induced toxicity and organ dysfunction, which are of great importance to the field of drug discovery.
Unfortunately, RNA content has been shown to be severely depleted during histochemical assays, for example, by immunostaining of tissue sections (Fink et al. (2000), Lab Invest 80:327–333; Kohda et al. (2000), Kidney Int 57:321–331). This has practically precluded mRNA expression analysis of immunostained tissue, either by in situ hybridization or by microarray gene profiling.
It was generally assumed that RNA in tissue sections was degraded by endogenous RNases during the immunostaining protocol (Murakami et al. (2000), Kidney Int 58:1346–1353). Therefore, to preserve RNA in the tissue section during an immunostaining, large amounts of RNase inhibitors (Murakami et al. (2000), supra) or various tissue fixatives such as formalin (Fink et al. (2000), supra) have been used in modified immunostaining protocols. See, e.g., U.S. Patent Application Publication No. US 2002/0009768. Although these protocols have had varying degrees of success, in general they have to be extremely short in duration (Fend et al. (1999), Am J Pathol 154:61–66). These modified immunostaining protocols have limited usefulness because a longer incubation period is required for the better sensitivity of immunostaining detection.
Accordingly, a method to robustly preserve RNA in a biological sample is needed to facilitate investigation of mRNA expression patterns within a specific cell population or tissue.